A micro total analysis system, operating method and manufacturing method thereof are provided. The micro total analysis system includes at least one micro total analysis unit each including: microfluidic device including first electrode and dielectric layer connected to each other, where the dielectric layer drives to-be-measured droplet to move based on voltage of the first electrode; and acoustic wave detection device including second electrode connected to the dielectric layer, where the dielectric layer is also used as transducer of the acoustic wave detection device, and configured to generate acoustic wave toward the droplet based on voltage of the second electrode, and generate a detection result corresponding to the droplet based on received acoustic wave. The micro total analysis system, the operating method and the manufacturing method thereof enables the microfluidic device and the acoustic wave detection device to be integrated in the same chip.